Project Summary The long term goal of our proposal is to understand the mechanisms by which transfusion of older packed red blood cell (pRBC) units worsens patient outcomes after resuscitation from hemorrhage. Hemorrhagic shock is the most common cause of potentially preventable death after trauma. Current resuscitation strategies for patients with significant blood loss include the use pRBCs and fresh-frozen plasma. While the use of pRBCs for resuscitation of the injured patient is essential for survival, transfusion of pRBC units that have aged during storage is associated with worsened clinical outcomes in patients, including increased risk of multisystem organ failure, pneumonia, renal failure, sepsis, and death. We have demonstrated that microparticles from aged pRBC units contain red blood cell microparticles that mediate inflammatory events after transfusion. Our previous studies and preliminary data strongly indicate that microparticle formation is a key event during pRBC storage and that pRBC microparticles are critical mediators of lung inflammation after resuscitation from hemorrhage. In the current proposal, we hypothesize that microparticles from stored packed red blood cells are acutely pro-inflammatory in nature and promote inflammatory consequences, such as endothelial cell activation, formation of pulmonary microthrombi, and development of lung inflammation after resuscitation from hemorrhage. To test this hypothesis, we propose the following specific aims: Aim 1: Test mechanistic strategies to reduce pRBC microparticle generation during storage and mitigate pro-inflammatory potential of stored pRBC units; Aim 2: Determine the molecular mechanisms of endothelial cell activation by microparticles from stored pRBC units; Aim 3: Determine the mechanisms by which pRBC microparticles promote multi-cellular interactions leading pulmonary microthrombi after hemorrhage and resuscitation. The proposed studies will general novel data concerning the role of microparticles from stored pRBC units and the development of endothelial cell dysfunction after hemorrhage and resuscitation. If successful, these studies will identify new therapeutic targets allowing improved clinical outcomes after hemorrhage and resuscitation with stored pRBC units.